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Vascular endothelial growth factor A (VEGF-A) is considered one of the most important factors in tumor angiogenesis, and consequently, a number of therapeutics have been developed to inhibit VEGF signaling. Therapeutic strategies to target brain malignancies, both primary brain tumors, particularly in pediatric patients, and metastases, are lacking, but targeting angiogenesis may be a promising approach. Multiparametric MRI was used to investigate the response of orthotopic SF188 luc pediatric glioblastoma xenografts to small molecule pan-VEGFR inhibitor cediranib and the effects of both cediranib and cross-reactive human/mouse anti-VEGF-A antibody B20-4.1.1 in intracranial MDA-MB-231 LM2-4 breast cancer xenografts over 48 hours. All therapeutic regimens resulted in significant tumor growth delay. In cediranib-treated SF188 luc tumors, this was associated with lower K trans (compound biomarker of perfusion and vascular permeability) than in vehicle-treated controls. Cediranib also induced significant reductions in both K trans and apparent diffusion coefficient (ADC) in MDA-MB-231 LM2-4 tumors associated with decreased histologically assessed perfusion. B20-4.1.1 treatment resulted in decreased K trans , but in the absence of a change in perfusion; a non-significant reduction in vascular permeability, assessed by Evans blue extravasation, was observed in treated tumors. The imaging responses of intracranial MDA-MB-231 LM2-4 tumors to VEGF/VEGFR pathway inhibitors with differing mechanisms of action are subtly different. We show that VEGF pathway blockade resulted in tumor growth retardation and inhibition of tumor vasculature in preclinical models of pediatric glioblastoma and breast cancer brain metastases, suggesting that multiparametric MRI can provide a powerful adjunct to accelerate the development of antiangiogenic therapies for use in these patient populations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5 mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5 mC clusters and a biased distribution of 5 mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5 mC in D-loop formation, rather than in mitochondrial gene expression., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Glutaraldehyde-fixed pericardium of animal origin is the elective material for the fabrication of bio-prosthetic valves for surgical replacement of insufficient/stenotic cardiac valves. However, the pericardial tissue employed to this aim undergoes severe calcification due to chronic inflammation resulting from a non-complete immunological compatibility of the animal-derived pericardial tissue resulting from failure to remove animal-derived xeno-antigens. In the mid/long-term, this leads to structural deterioration, mechanical failure, and prosthesis leaflets rupture, with consequent need for re-intervention. In the search for novel procedures to maximize biological compatibility of the pericardial tissue into immunocompetent background, we have recently devised a procedure to decellularize the human pericardium as an alternative to fixation with aldehydes. In the present contribution, we used this procedure to derive sheets of decellularized pig pericardium. The decellularized tissue was first tested for the presence of 1,3 α-galactose (αGal), one of the main xenoantigens involved in prosthetic valve rejection, as well as for mechanical tensile behavior and distensibility, and finally seeded with pig- and human-derived aortic valve interstitial cells. We demonstrate that the decellularization procedure removed the αGAL antigen, maintained the mechanical characteristics of the native pig pericardium, and ensured an efficient surface colonization of the tissue by animal- and human-derived aortic valve interstitial cells. This establishes, for the first time, the feasibility of fixative-free pericardial tissue seeding with valve competent cells for derivation of tissue engineered heart valve leaflets., (© 2015 Wiley Periodicals, Inc.)

Epigenetics harbours all regulatory information that, beyond nucleotide sequences, allows cells to "make decisions" throughout their lifetime in response to the external environment. The information can be transitory or relatively stable, and is even transmittable either to daughter cells or to the next generations through the germ line. Recent discoveries shed light on numerous connections between metabolites and epigenetic chromatin-modifying enzymes, providing a link between the metabolic state of the cell and epigenetics, and ultimately between metabolism, gene expression and cell fate. In this review, we discuss the possible connections between metabolism and epigenetic regulation of stem cell differentiation and self-renewal. Moreover, we describe pertinent literature that could explain how altered metabolic state and nutrition can contribute to disease development through epigenetic modifications. A special section is dedicated to the emerging link between the circadian clock, metabolic transcriptional regulation by epigenetic mechanisms and their implication in stem cell homeostasis.

The function of human circulating PACs has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines and then exposed to proinflammatory cytokines or oxLDL. Under these conditions, PACs converted into APCs, expressed maturation markers CD80 and CD83, and showed an increased up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters," showing reciprocal modulation in APCcy vs. APCox, justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotypes and functions into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions., (© Society for Leukocyte Biology.)

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck - SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.

Hepatitis B is a major infectious occupational hazard for health care workers and can be prevented with a safe and effective vaccine. The serum titer of anti-HBsAg antibodies is the most commonly used correlate of protection and post-vaccination anti-HBsAg concentrations of ≥ 10 mIU/ml are considered protective. Subjects with post-vaccination anti-HBsAg titers of <10 mIU/ml 1-6 months post-vaccination, who tested negative for HBsAg and anti-HBc, are defined as non-responders. The question of whether non-responders should be repeatedly vaccinated is still open. The aim of the study was to (i) evaluate the distribution of lymphocyte subpopulations and the percentage of HBsAg-specific memory B cells in responders and non-responders (ii) assess whether non-responders can be induced to produce antibodies after administration of a booster dose of vaccine (iii) determine whether booster vaccination increases the number of specific memory B cells in non-responders. Combining flow-cytometry, ELISPOT and serology we tested the integrity and function of the immune system in 24 health care workers, confirmed to be non-responders after at least three vaccine injections. We compared the results with those obtained in 21 responders working in the same institution. We found that the great majority of the non-responders had a functional immune system and a preserved ability to respond to other conventional antigens. Our most important findings are that the frequency of HBsAg-specific memory B cells is comparable in non-responders and controls and that booster immunization does not lead either to antibody production or memory B cell increase in non-responders., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Whole-genome sequencing studies have recently identified a quarter of cases of the rare childhood brainstem tumor diffuse intrinsic pontine glioma to harbor somatic mutations in ACVR1. This gene encodes the type I bone morphogenic protein receptor ALK2, with the residues affected identical to those that, when mutated in the germline, give rise to the congenital malformation syndrome fibrodysplasia ossificans progressiva (FOP), resulting in the transformation of soft tissue into bone. This unexpected link points toward the importance of developmental biology processes in tumorigenesis and provides an extensive experience in mechanistic understanding and drug development hard-won by FOP researchers to pediatric neurooncology. Here, we review the literature in both fields and identify potential areas for collaboration and rapid advancement for patients of both diseases., (©2014 American Association for Cancer Research.)

Surgical flow augmentation for treatment of cerebral hemodynamic impairment remains controversial. Here, we investigated the benefit of endothelial progenitor cell (EPC) treatment in a rat model of chronic cerebral hypoperfusion. At repeated time points after 3-vessel occlusion (3-VO), animals were treated with 1 × 10(6) DiI-labeled (a) ex vivo-expanded embryonic-EPC (e-EPC), (b) cyclic AMP-differentiated embryonic-endothelial progenitor-derived cells (e-EPDC as biologic control) or, (c) saline. The cerebrovascular reserve capacity (CVRC) was assessed immediately before and on days 7 and 21 after 3-VO. Structural effects were assessed by latex perfusion, immunohistochemistry, and intravital fluorescence video microscopy on day 21. Three-vessel occlusion resulted in a significant impairment of the CVRC with better functional recovery after treatment with e-EPC (16.4±8%) compared with e-EPDC (3.7±8%) or saline (6.4±9%) by day 21 (P<0.05), which was paralleled by a significant increase in the vessel diameters of the anterior Circle of Willis, a significantly higher number of leptomeningeal anastomoses and higher parenchymal capillary density in e-EPC-treated animals. Interestingly, despite in vivo interaction of e-EPC with the cerebral endothelium, e-EPC incorporation into the cerebral vasculature was not observed. Our results suggest that EPC may serve as a novel therapeutic agent in clinical trials for nonsurgical treatment of chronic cerebral hemodynamic impairment.

Background: Several studies have shown a higher risk of psychological problems in health care workers exposed to serious occupational stressors., Objectives: The aim of the present study was to assess the presence of Post-traumatic Stress Disorder (PTSD) and possible risk factors in a sample of 32 workers who were at the same time rescuers and victims of a fire that broke out in the neonatal intensive care unit of a large paediatric hospital., Methods: Immediately and six months after the event, the subjects underwent a study protocol aimed at the diagnostic assessment of PTSD, investigated via the Clinician-Administered PTSD Scale (CAPS) and the Impact of Event Scale-Revised (IES-R)., Results: Out of the 30 subjects examined (two were missing), six showed the diagnostic criteria for a current PTSD. Risk factors for PTSD onset were a prior psychiatric disorder, the level of involvement in the fire disaster and the presence of phobias in the days immediately after the event. Gender and level of education approached statistical significance., Conclusions: The high prevalence of PTSD found in this sample was due to the fact that the risk of death or serious injury involved infants.

Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9-12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH 4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.

Background Aims: Endothelial progenitor cells (EPCs) specifically home to sites of malignant growth, rendering them attractive for anti-cancer therapies. Data are conflicting on the phenotype and quantitative contribution toward tumor angiogenesis based on differing culture assays to outgrow EPCs. To evaluate the origin and early phenotype of EPCs and to define a population with enhanced tumor-targeting capacity, we evaluated a hierarchy of cord blood-derived EPCs modeling the multi-step nature of tumor homing., Methods: CD34(+) mononuclear cells were isolated from fresh cord blood and cultured to derive endothelial colony-forming cells (ECFCs). Human umbilical vein endothelial cells (HUVECs) served as control. Using intra-vital microscopy, the recruitment was analyzed in mice bearing C6 xenografts. Adhesion, migration, transmigration and differentiation were further addressed., Results: Within the primary passage, ECFCs underwent a rapid maturation from a CD45(+) and CD31(+) phenotype to a CD45(-) and endothelial marker positive phenotype. Assessing in vivo tumor recruitment, ECFCs had the highest activity in all steps analyzed. In vitro, ECFCs demonstrated significantly higher adhesion under static and flow conditions. Similarly, ECFCs exhibited highest migratory and trans-migratory activity toward tumor-conditioned medium. On subcutaneous implantation, only ECFCs formed blood vessels covered with perivascular cells, similar to HUVECs., Conclusions: Our study indicates that ECFCs emerge from a CD45(+) and CD31(+) progenitor and rapidly mature in culture. ECFCs have a significantly higher potential for tumor targeting than non-cultured CD34(+) cells and HUVECs. They are ideal candidates for future cell-based anti-cancer therapies., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Epigenetics, through control of gene expression circuitries, plays important roles in various physiological processes such as stem cell differentiation and self renewal. This occurs during embryonic development, in different tissues, and in response to environmental stimuli. The language of epigenetic program is based on specific covalent modifications of DNA and chromatin. Thus, in addition to the individual identity, encoded by sequence of the four bases of the DNA, there is a cell type identity characterized by its positioning in the epigenetic "landscape". Aberrant changes in epigenetic marks induced by environmental cues may contribute to the development of abnormal phenotypes associated with different human diseases such as cancer, neurological disorders and inflammation. Most of the epigenetic studies have focused on embryonic development and cancer biology, while little has been done to explore the role of epigenetic mechanisms in the pathogenesis of cardiovascular disease. This review highlights our current knowledge of epigenetic gene regulation and the evidence that chromatin remodeling and histone modifications play key roles in the pathogenesis of cardiovascular disease through (re)programming of cardiovascular (stem) cells commitment, identity and function.

Background: The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue., Principal Findings: The aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice., Conclusions: Our results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.

Unlabelled: Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcription—K27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein., Significance: We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies

One undisputed milestone of traditional oncology is neoplastic progression, which consists of a progressive selection of dedifferentiated cells driven by a chance sequence of genetic mutations. Recently it has been demonstrated that the overexpression of well-defined transcription factors reprograms somatic cells to the pluripotent stem status. The demonstration raises crucial questions as to whether and to what extent this reprogramming contributes to tumorigenesis, and whether the epigenetic changes involved in it are reversible. Here, we show for the first time that a tumor produced in vivo by a chemical carcinogen is the product of the interaction between neoplastic progression and reprogramming. The experimental model employed the prototype of ascites tumors, the Yoshida AH130 hepatoma and other neoplasias, including human melanoma. AH130 hepatoma was started in the liver by the carcinogen o-aminoazotoluene. This compound binds to and abolishes the p53 protein, producing a genomic instability that promotes both the neoplastic progression and the hepatoma reprogramming. Eventually this tumor contained 100% CD133(+) elements and pO(2)-dependent percentages of the three embryonic transcription factors Nanog, Klf4 and c-Myc. Once transferred into aerobic cultures, the minor cellular fraction expressing this triad generates various types of adherent cells, which are progressively substituted by non-tumorigenic elements committed to fibromuscular, neuronal and glial differentiation. This reprogramming appears to be accomplished stepwise, with the assembly of the triad into a sophisticated transcriptional, oxygen-dependent circuitry, in which Nanog and Klf4 antagonistically regulate c-Myc, and hence, cell hypoxia survival and cell cycle activation.

Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), homogeneous cultures that do not take into account tumor heterogeneity, three-dimensional (3D) cell-cell contacts between tumor and/or host cells or interactions with extracellular matrix proteins. Here we describe a 3D tumor spheroid-based migration assay which more accurately reflects the solid tumor microenvironment and can accommodate both extracellular matrix and host cell interactions. It is a rapid and highly reproducible 96-well plate-based technique and we demonstrate its utility for the evaluation of therapeutic agents/drugs with anti-migratory properties.

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment., (Copyright © 2012 AlphaMed Press.)

Ultraviolet radiation is known to cause both benefits and harmful effects on humans. The adverse effects mainly involve two target organs, skin and eye, and can be further divided into short- and long-term effects. The present case report describes an accidental exposure of two health-care workers to ultraviolet radiation produced by a germicidal lamp in a hospital pharmacy. The germicidal lamp presented a spectrum with an intense UV-C component as well as a modest UV-B contribution. Overexposure to UV-C radiation was over 100 times as large as the ICNIRP exposure limits. A few hours after the exposure, the two subjects reported symptoms of acute UV injury and both of them continued having significant clinical signs for over 2 years. In this study, we describe acute and potentially irreversible effects caused by high UV exposure. In addition, we present the results of risk assessment by occupational exposure to germicidal lamps., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Background: There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed., Results: To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease., Conclusions: We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Background: Assessing the quality of care and service delivery through the analysis of ad hoc indicators is a relevant process for quality monitoring and improvement with a view to providing patients, the community and administrators alike with appropriate elements of evaluation. This paper describes the key results of a certification process based on the Joint Commission International (JCI) criteria for the home hospitalisation model implemented by the palliative care units of seven Milan hospitals for terminally ill patients with cancer., Methods: In 2006, the interhospital working team implemented a certification project based on the JCI Disease or Condition-Specific Care (DSCS) programme. Thirty standards subdivided into five functional areas with 150 measurable elements were the starting-point for periodic improvement plans within and across participating hospitals. Programme compliance was analysed in terms of annual performance improvement and consistency across the seven PCUs involved in achieving set goals. The JCI standards were applied on 3316 terminally ill patients with cancer treated at home from 2005 to 2009., Results: As a result of the work carried out, the JCI survey conducted 3 years after project implementation demonstrated full compliance with the established standards, leading to the JCI certification award (for the first time in this clinical setting internationally)., Conclusion: The work carried out with a view to certification has confirmed the possibility that facilities spread across different hospitals can actually share common processes and standardise the activities for the care of end-of-life patients with cancer at home as if they were one single service provider.

Background: Assessing the quality of care and service delivery through the analysis of ad hoc indicators is a relevant process for quality monitoring and improvement with a view to providing patients, the community and administrators alike with appropriate elements of evaluation. This paper describes the key results of a certification process based on the Joint Commission International (JCI) criteria for the home hospitalisation model implemented by the palliative care units of seven Milan hospitals for terminally ill patients with cancer., Methods: In 2006, the interhospital working team implemented a certification project based on the JCI Disease or Condition-Specific Care (DSCS) programme. Thirty standards subdivided into five functional areas with 150 measurable elements were the starting-point for periodic improvement plans within and across participating hospitals. Programme compliance was analysed in terms of annual performance improvement and consistency across the seven PCUs involved in achieving set goals. The JCI standards were applied on 3316 terminally ill patients with cancer treated at home from 2005 to 2009., Results: As a result of the work carried out, the JCI survey conducted 3&emsp14;years after project implementation demonstrated full compliance with the established standards, leading to the JCI certification award (for the first time in this clinical setting internationally)., Conclusion: The work carried out with a view to certification has confirmed the possibility that facilities spread across different hospitals can actually share common processes and standardise the activities for the care of end-of-life patients with cancer at home as if they were one single service provider.

Recently, carbonic anhydrase (CA) inhibitors have been proposed as a potential new class of antitumor agents. The aim of this study was to evaluate the antitumor activity of three CA inhibitors, namely acetazolamide (AZ) and two newly synthesized aromatic sulfonamides with high affinity for CA IX, 2-(4-sulfamoylphenyl-amino)-4,6-dichloro-1,3,5-triazine (TR1) and 4-[3-(N,N-dimethylaminopropyl)thioreidophenylsulfonylaminoethyl]benzenesulfonamide (GA15), against human tumor cells. The effects of AZ, TR1, and GA15 on cell proliferation and apoptosis were evaluated in CA IX-positive HeLa and 786-O cells and CA IX-negative 786-O/von Hippel-Lindau (VHL) cells. We also investigated whether the potential antitumor activity of these molecules might be mediated by an increase in ceramide production. AZ, TR1, and GA15 could significantly reduce cell proliferation and induce apoptosis in HeLa and 786-O cells. Moreover, all three inhibitors could decrease intracellular pH (pH(i)) and increase ceramide production in the same cells. Treatment with the ceramide synthase inhibitor fumonisin B1 prevented the apoptotic effects of the three CA inhibitors. In all experiments, the effects of aromatic sulfonamides were more pronounced than those of AZ. The three inhibitors did not show any antitumor activity in CA IX-negative 786-O/VHL cells and failed to lower pH(i) or increase intracellular ceramide levels in the same cells. In conclusion, CA inhibition can decrease cell proliferation and induce apoptosis in human tumor cells. The ability of CA inhibitors to decrease pH(i) might trigger cell apoptosis through mediation of ceramide synthesis. Activation of this apoptotic cascade probably is mediated by inhibition of the CA IX isoform.

A genome-wide association screen for primary biliary cirrhosis risk alleles was performed in an Italian cohort. The results from the Italian cohort replicated IL12A and IL12RB associations, and a combined meta-analysis using a Canadian dataset identified newly associated loci at SPIB (P = 7.9 x 10(-11), odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 x 10(-10), OR = 1.63) and 17q12-21 (P = 1.7 x 10(-10), OR = 1.38).

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-alpha and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-alpha, IL-1beta, IL-6, and IL-10 were released in wild-type and TLR2(-/-), but not TLR4(-/-), mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.

Neovascularization can be improved using polymer scaffolds supporting endothelial progenitor cells (EPCs). The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured in a hyaluronan-based non-woven mesh (HYAFF-11). eEPCs were seeded on HYAFF-11 at the density of 1 x 10(6)/cm(2) and cultured with endothelial differentiating factors for 3 weeks. After 24 h, nearly 90% of EPCs were adherent. Cell viability, evaluated by methyltetrazolium test, was greater in HYAFF-11 than on the most commonly used fibronectin-coated dishes, even if a progressive decline in viability was observed starting from approximately the second week of culture. eEPCs easily migrated to and aggregated on the scaffold. Evidence of active protein synthesis and features of endothelial differentiation, including cellular transcytotic channels and micropinocytotic vesicles, was revealed using electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction analysis. eEPCs cultured in the scaffold also showed a certain angiogenic activity, as demonstrated by hepatocyte growth factor transcription and vascular endothelial growth factor secretion. In conclusion, eEPCs can migrate and adhere inside HYAFF-11, maintain their pre-endothelial phenotype, and express angiogenic factors, especially within the first week of growth. These results indicate that non-woven HYAFF-11 could be a promising candidate as a vehicle for eEPCs for regenerative medicine applications.

Purpose: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB1 and CB2, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation., Experimental Design: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB1 agonist arachinodyl-2'-chloroethylamide and the CB2 agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-alpha production were evaluated. The knockdown of TNF-alpha mRNA was obtained with the use of selective small interfering RNA., Results: We show that the CB1 receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB2 receptor. The activation of the CB1 and, more efficiently, of the CB2 receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB2 agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-alpha mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation., Conclusions: The present study shows that either CB1 or CB2 receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-alpha acts as a link between cannabinoid receptor activation and ceramide production.

Circulating endothelial progenitor cells (EPCs) can contribute to neovascularization, even if the mechanisms by which they interact with mature endothelial cells remain unclear. The interactions between human coronary artery endothelial cells (HCAECs) and peripheral blood mononuclear cells (PBMCs) during their early differentiation towards an EPC phenotype were investigated. A co-culture model, in which the two cell types share the same culture medium in the absence of any exogenous angiogenic stimulus, was used. The role of hypoxia was assessed by pretreating HCAECs with 3% O(2) before co-culture setting. Since we have previously shown that both adherent and suspended PBMCs display a significant increase in endothelial marker expression within the 2nd day of culture in an angiogenic environment, the role of HCAECs on early PBMC differentiation was evaluated in both adherent and suspended cell fractions. A 3-day co-culture period increased the expression of VEGF-R2, VE-cadherin, alpha(v)beta(3)- and alpha(5)-integrin in both the adherent and suspended PBMCs, assessed by cytofluorimetric analysis, and up-regulated VEGF-R1 mRNA assessed by real-time RT-PCR. HCAECs influenced PBMC adhesion, transendothelial migration and cell organization on Matrigel. Hypoxia modulated either PBMC differentiation or their functional properties. These data strongly suggest that endothelium may support the differentiation of PBMCs into EPCs.

We hypothesized that circulating polymorphonuclear granulocytes (PMNs), vascular endothelial cells (ECs), and perivascular mast cells (MCs) may initiate and sustain the inflammatory response through the generation of the superoxide anion (O(2)(*-)) by PMNs primed by inflammatory stimuli, which in turn evoked the overexpression of adhesion molecules from ECs and release of histamine by MCs. To pin-point the role of carbon monoxide (CO) in curbing vascular inflammation, we studied the effect of a water-soluble CO-releasing molecule [tricarbonylchloro-glycinate-ruthenium (II); CORM-3] on an experimental model of vascular inflammation. The model consists of coincubating formyl-methionyl peptide (fMLP) -primed human PMNs with rat ECs or with rat MCs. The effects of CORM-3 were evaluated by measuring the generation of O(2)(*-) and the expression of CD11b in fMLP-primed PMNs; the expression of ICAM-1 and CD203c in ECs and MCs, respectively; and the release of histamine from MCs. Our results show that the chemotactic peptide fMLP primes PMNs to generate O(2)(*-) and overexpress CD11b, both events being central to the inflammatory process, while CORM-3 significantly decreases these events (IC(50)=1.66 microM for O(2)(*-) production; 1.20 microM for CD11b expression in human PMNs). The experiments also show that fMLP-primed PMNs increase the CD54 expression by coincubated ECs, and the expression of CD203c and the release of histamine by coincubated MCs. Once again, CORM-3 abolishes these events (IC(50)=6.78 microM for CD54 expression in ECs; 1.18 microM for CD203 expression; 1.15 microM for histamine release in MCs). Thus, CORM-3 exerts a powerful anti-inflammatory action by down-regulating the oxidative burst in PMNs, the overexpression of adhesion molecules in PMNs and ECs, the release of histamine, and the overexpression of an activation marker by MCs.

Bartonella quintana (B. quintana) is a facultative, intracellular bacterium, which causes trench fever, chronic bacteraemia and bacillary angiomatosis. Little is known about the recognition of B. quintana by the innate immune system. In this review, we address the impact of Toll-like receptors (TLRs) on the recognition of B. quintana and the activation of the host defense. When experimental models using human mononuclear cells, transfected CHO cells, or TLR2-/- and TLR4-/- mice were used, differential effects of TLR2 and TLR4 have been observed. B. quintana micro-organisms stimulated cytokine production through TLR2-mediated signals, whereas no role for TLR4 in the recognition of this pathogen was observed. When single, water-phenol extraction was performed, B. quintana LPS, stimulated cytokine production in a TLR2-dependent manner. However, when double extraction was performed in order to generate highly purified LPS, B. quintana LPS entirely lost its capacity to stimulate cytokines, demonstrating that non-LPS components of B. quintana are responsible for the recognition through TLR2. Moreover, B. quintana LPS was shown to be a potent antagonist of Toll-like receptor 4 (TLR4). In conclusion, B. quintana is an inducer of cytokines through TLR2-, but not TLR4-, dependent mechanisms. This stimulation is induced by bacterial components other than lipopolysaccharide. B. quintana LPS is a naturally occurring antagonist of Toll-like receptor 4 (TLR4). In view of the role played by TLR4 in inflammation, B. quintana LPS may be useful as an anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

Aim: To assess the safety and efficacy of percutaneous radiofrequency thermal ablation (RFA) in the treatment of nonsurgical hepatocellular carcinoma (HCC) in daily practice., Methods: A total of 63 consecutive patients with HCC (solitary nodule

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.

Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against beta1 and beta2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to beta2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing.

Background: The pendulum test of Wartenberg is a technique commonly used to measure passive knee motion with the aim to assess spasticity. We used this test to evaluate changes of the knee angular displacement, passive stiffness and viscosity in rheumatoid arthritis patients. Stiffness and viscosity represent passive resistances to joint motion associated with the structural properties of the joint tissue and of the muscular-tendon complex. Stiffness can be considered an intrinsic property of the tissues to resist deformation, while viscosity is related to cohesive forces between adjacent layers of tissues. Both parameters may influence the joint range of motion affecting angular displacement., Methods: Nine women with rheumatoid arthritis were compared with a group of healthy women. With the subject half-lying, the relaxed knee was dropped from near-full extension and the characteristics of the ensuring damped unsustained knee oscillation evaluated. The kinematics of leg oscillations was recorded using ultrasonic markers (Zebris CMS HS 10) and the kinetic data were calculated from kinematic and anthropometric measures., Results: Knee stiffness significantly increased (p < 0.001) in patients with respect to the control group, while differences in viscosity were not significant. Moreover, the amplitudes of first knee flexion (the maximal flexion excursion after knee release) and first knee extension (the maximal extension excursion after the first knee flexion) were significantly decreased (p < 0.001). A regression analysis showed that disease severity correlated moderately with stiffness (R2 = 0.68) and first flexion (R2 = 0.78). Using a multivariate regression, we found that increasing stiffness was the main factor for the reduction of flexion and extension motions., Conclusion: We showed that the Wartenberg test can be considered a practical tool to measure mechanical changes of knee caused by rheumatoid arthritis. This novel application of Wartenberg test could be useful to follow up the effects of pharmacological and rehabilitative interventions in this disease.

Conflicting data exist regarding the relationship between hepatitis C virus genotype 1 and hepatic steatosis as well as the latter's role in the progression of fibrosis and treatment response. We assessed factors associated with hepatic steatosis in genotype 1 chronic hepatitis C and the impact of hepatic fat on fibrosis development and interferon responsiveness. Two hundred ninety-one non-diabetic patients with genotype 1 chronic hepatitis C were examined for the presence of steatosis and its correlation with clinical, virological, and biochemical data, including insulin resistance (IR), evaluated by the homeostasis model assessment (HOMA) score. Steatosis was graded as mild (1%-20% of hepatocytes involved), moderate (21%-40% of hepatocytes involved), and severe (>40% of hepatocytes involved). Steatosis was mild in 110 of 291 (37.8%) and moderate/severe in 55 of 291 (18.9%) subjects. By logistic regression, moderate/severe steatosis was independently associated with the female sex (odds ratio [OR] 2.74; 95% CI 1.40-5.35), high gamma-glutamyltransferase levels (OR 1.52; 95% CI 1.22-1.91), and HOMA-score (OR 1.076; 95% CI 1.001-1.26). By logistic regression, moderate/severe steatosis (OR 2.78; 95% CI 1.21-6.4), and platelet counts (OR 0.97; 95% CI 0.96-0.98) were independent predictors of advanced fibrosis. Patients with moderate/severe steatosis had an OR of 0.52 (95% CI 0.30-0.90) for sustained virological response compared with patients with mild/absent steatosis. In conclusion, in nondiabetic European patients with genotype 1 hepatitis C at low risk for the metabolic syndrome, the prevalence of steatosis was nearly 60%. IR is a risk factor for moderate/severe steatosis, especially in men. Moderate/severe steatosis has clinical relevance, being associated with advanced fibrosis and hyporesponsiveness to antiviral therapy.

We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (PlGF) through the induction of functional fms-like tyrosine kinase (Flt-1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the PlGF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, PlGF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia-treated VSMCs in response to PlGF was significantly impaired by the p38 and the phosphatidylinositol 3-kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of PlGF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt-1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen-activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to PlGF, while the p38 inhibitor SB202190 markedly impaired the PlGF-induced contractile response. These effects were selectively mediated by Flt-1 without any involvement of foetal liver kinase-1 receptors. These data are the first evidence that different intracellular pathways activated by Flt-1 receptor in VSMCs are involved in diverse biological effects of PlGF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase(1/2) and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of PlGF in vascular pathology.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. Here we report that JAM-A is required for the correct infiltration of polymorphonuclear leukocytes (PMN) into an inflamed peritoneum or in the heart upon ischemia-reperfusion injury. The defect was not observed in mice with an endothelium-restricted deficiency of the protein but was still detectable in mice transplanted with bone marrow from JAM-A(-/-) donors. Microscopic examination of mesenteric and heart microvasculature of JAM-A(-/-) mice showed high numbers of PMN adherent on the endothelium or entrapped between endothelial cells and the basement membrane. In vitro, in the absence of JAM-A, PMN adhered more efficiently to endothelial cells and basement membrane proteins, and their polarized movement was strongly reduced. This paper describes a nonredundant role of JAM-A in controlling PMN diapedesis through the vessel wall.

Background/aims: We assessed the effectiveness and safety of an induction dose of peginterferon alfa-2b (PEG-IFN) plus ribavirin for initial treatment of patients with genotype 1 chronic HCV infection in a randomized, controlled, multicenter trial., Methods: Three hundred and eleven naïve patients infected with genotype 1 and chronic hepatitis were randomly assigned to 48-week treatment with PEG-IFN once weekly (80-100 micrograms depending on body weight for 8 weeks, followed by 50 micrograms for the next 40 weeks), or standard interferon alfa-2b (IFN) 6 million units on alternate days, both in combination with ribavirin (1000-1200 mg/day)., Results: PEG-IFN plus ribavirin significantly increased sustained virological response (SVR) compared with IFN plus ribavirin (41.1 vs. 29.3% respectively, P=0.030). Less patients discontinued PEG-IFN than IFN (19 vs. 31%, P=0.010). By logistic regression, SVR in the PEG-IFN group was independently associated with age <50 years, and mild fibrosis at liver biopsy., Conclusions: Combination therapy with an induction dose of PEG-IFN was a more effective and better tolerated treatment for naïve patients with genotype 1 than combination therapy with high dose standard IFN. In patients aged less than 50 years with mild fibrosis this schedule achieves a very high rate of SVR.

To study the effect of growth factors on iatrogenic apoptosis, we examined the influence of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on staurosporine-induced apoptosis in primary cultures of human umbilical vein endothelial cells (HUVEC). Apoptosis was evaluated by a cell viability test, the TUNEL-POD assay and the activation of the pro-apoptotic caspase-3. Staurosporine (10-100nM) caused the activation of caspase-3. This effect was manifest after 2hr of incubation and reached its maximum after 5hr. Severe loss of viability followed within 18hr. VEGF or EGF (10-100ng/mL) added together with staurosporine decreased the activation of caspase-3. The loss of viability was 24hr delayed. The action of growth factors was observed at 1% serum concentration but also at concentration optimal for HUVEC survival (10%, v/v). Furthermore, the inhibition of PI-3 kinase (PI-3K) by wortmannin or LY294002 as well as the inhibition of MEK by PD098059 or U0126 prevented the protective effect of VEGF and EGF. Western blotting analysis showed that after 3hr of incubation with staurosporine the level of the anti-apoptotic protein Mcl-1 decreased and this effect was reverted by VEGF. It is concluded that VEGF and EGF antagonize the pro-apoptotic action of staurosporine by the combined signalling of PI-3K and ERKs pathways.